Parkinsonia aculeata suppresses inflammation and cartilage destruction in collagen-induced arthritic rat

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M G. Gavaniya*1, N L. Pathak1, H P. Trivedi1, A K. Patel1, Dr. H D. Trivedi1, N M. Panchal1, Dr L D. Patel1
1Department of Pharmacology,

C U Shah College of Pharmacy and Research,

Wadhwan, Gujarat-363030.

*For correspondence

Mahendra G Gavaniya,


Ta- Chuda

Dist- Surendranagar-363415

E- mail:


Parkinsonia aculeata (P. aculeata) has multiple applications in Indian traditional medicine because of its anti-pyretic, anti-inflammatory, antidiabetic and antimicrobial activities and has long been known to have anti-inflammatory effects. However, no study on the anti-arthritic activity of Parkinsonia aculeata has been reported in vivo. Rheumatoid arthritis (RA) is a systemic autoimmune disease with chronic inflammation characterized by hyperplasia of synovial cells in affected joints, which ultimately leads to the destruction of cartilage and bone. We investigated therapeutic efficacy of Parkinsonia aculeata in treating Rheumatoid Arthritis (RA) using collagen-induced arthritis (CIA) animal model. Arthritis was induced in wistar rats by immunization with bovine type II collagen. CIA rats were treated daily with oral administration of different doses of Methanolic extract of P. aculeata (MPA) beginning on the day after the onset of arthritis (day 21st, the therapeutic treatment) until day 45. The results showed that treatment with MPA markedly reduced paw swelling and arthritic index even in the established CIA. Radiologic and histopathologic changes in the arthritic joints were also significantly reduced in the MPA-treated versus vehicle-treated rats. Moreover, Rheumatoid factor was significantly reduced in MPA treated group in compared to disease control group. Hence, our studies demonstrate safety, and effectiveness of P. aculeata as an anti-arthritic agent, which makes P. aculeata a strong candidate for further research on rheumatoid arthritis (RA).

KEY WORDS: Collagen type –II induce arthritis, Parkisonia aculeata, Bone erosion


Rheumatoid arthritis (RA) is a chronic inflammatory disease involving multiple joints. The main pathology of the affected synovial tissue consists of hyperplasia and subintimal infiltration of T and B lymphocytes. Synovial tissue hyperplasia forms the pannus tissue that irreversibly destroys the cartilage and bone in the affected joint. Collagen-induced arthritis (CIA) rat is a widely studied animal model of inflammatory polyarthritis with similarities to RA. CIA is induced after immunization of susceptible strains of rat with bovine type II collagen (CII) in Incomplete Freund’s adjuvant (IFA), and the resulting disease is primarily mediated by an autoimmune response. [1,2] The significance of the model lies in the fact that CII is the major constituent protein of the cartilage in the diarthrodial joints—the primary site affected in RA.[2]

Drug therapy for RA is based on two principal approaches: symptomatic treatment with non-steroidal anti-inflammatory drugs (NSAIDs) and disease-modifying antirheumatic drugs (DMARDs).[3] However, most of the currently available drugs are primarily directed towards the control of pain and/or the inflammation associated with joint synovitis, but do little to interfere with the underlying immune-inflammatory events, and consequently also do little to block the disease progression and reduce cartilage and bone destruction of joints. A systematic review of randomized placebo-controlled trials conducted recently demonstrates that the published evidence supports only the efficacies of nine agents in Western medicine, i.e., infliximab, cyclosporin, sulphasalzine, leflunomide, methotrexate, parenteral gold, corticosteroids, auranofin and IL-1 receptor antagonist, in decreasing radiological progression in RA.[4] Nevertheless, a variety of problems exist with these drugs. For example, the use of methotrexate and leflunomide is impeded by their long-term side-effects and toxicity,[5] while cytokines antagonists, despite substantial efficacy and clinical improvement, entail high cost and incur hypersensitivity to medications and infections.[6] Consequently, there is dramatically growing interest in herbal medicines among persons with RA and in the RA research community.[7] In fact, herbal medicine is being widely used virtually around the world for treatment of rheumatic and arthritic diseases.[7,8,9,10,11] Thus, herbal medicines constitute a potentially important avenue leading to novel therapeutic agents for RA that may not only prevent structural damage of arthritic joints caused by tissue and bone breakdown, but also be safe, relatively inexpensive, highly tolerated, and convenient for many patients.

Parkinsonia aculeata is a small spiny tree, 4 – 10 m in height, with a short and often crooked trunk up to 40 cm in diameter, often branching near the ground with a very open crown of spreading branches. The plant is native of tropical America, found almost throughout the drier parts of India.[12] The various part of the plant is used for treatment of various diseases like, the infusion of dried flowers of Parkinsonia aculeata used orally as an antipyretic, abortifacient, diaphoretic in human adults.[13] Hot water extract of leaves used orally as abortifacient in pregnant women. It cures boils and tumors if young twigs are crushed and applied. Leaf, fruit and stem decoction are taken orally to treat fever, malaria. Many pharmacological activities viz. CNS depressant activity of ethanol-water (1:1) extract of dried aerial part, Smooth muscle stimulant activity of aqueous extract of dried aerial part of Parkinsonia aculeata,[14] Antibacterial activity taking ethanol-water extract of dried leaf of Parkinsonia aculeata,[15] Antidiabetic effect taking water soluble fraction of aerial parts of Parkinsonia aculeata have been reported earlier.[16]

Plant Material:

The disease free fresh plant material (Leaves) was collected in the month of September 2009 from surrounding area of Wadhwan, Gujarat and authenticated at Botany Department of Gujarat University by Dr. H. A. Solanki. After authentication, fresh leaves were collected in bulk from the tree, shade dried, pulverized and passed through sieve no.40 to obtain coarse powder.

Preparation of the Extract:

The powdered leaves (400 gm.) were subjected to continuous hot extraction with methanol in Soxhlet extractor for 48 hrs, followed by concentrating extract under vacuum. The extracts were stored in airtight bottle until use.

Phytochemical Studies:

The Methanolic extract of Parkinsonia aculeata (MPA) was subjected to phytochemical analysis. Methanolic extract showed the presence of alkaloids, flavonoids, saponins, carbohydrates, and terpenoids.


Protocol of the study was passed by Institutional Ethics Committee of C. U Shah College of Pharmacy and Research, Wadhwan. The study was carried out with adult female Wistar rats weighing 180–250 g. Animals were acclimatized to the experimental conditions in cages and kept under standard environmental conditions (22 ± 3◦C; 12/12 h light/dark cycle). Rats were allowed to feed and water ad libitum.

Induction of CIA and MPA treatment:

Arthritis was induced in female Wistar rats using the method described previously.[17] Collagen was dissolved overnight at 4°C in 0.1M Acetic acid to prepare concentration 2mg/ml. This solution was added dropwise to an equal volume of chilled incomplete freund’s adjuvant (IFA). On day first, animals (five groups, each containing six animals) receive total 0.5 ml collagen in 0.5 ml, equally divided in 5 sites. All injections are intradermal, one at the base of each appendages and one in the nape of neck. Seven days post-immunization, the animals receive identical booster injections. Control animals receive only the incomplete Freund’s adjuvant diluted with 0.1 M acetic acid. In the therapeutic treatment protocol for the established CIA, treatment with MPA, Dexamethazone, and vehicle were initiated on the day after the onset of arthritis (day 21) and continued once daily until day 45 of the experiment. Rats were treated orally with different dose of MPA (200, 400, 600 mg/kg of body weight), Dexamethazone (1mg/kg) and vehical until day 45 of experiment.

Evaluation of the development of arthritis:

Rats were inspected daily for the onset of arthritis characterized by edema and/or erythema in the paws. The incidence and severity of arthritis were evaluated using a system of arthritic scoring, and measurement of bi-hind paw volumes every 2 or 3 days beginning on the day when arthritic signs were first visible. Animals were observed for presence or absence of nodules in different organs like ear, fore paw, hind paw, nose and tail. Animal were score 0 for absence and 1 for presence of nodules. 5 was the potential maximum of combined arthritic score per animal. Hind paw volume was measured using plethismometer. Paw volumes of both hind limbs were recorded from 21st day to 45th day at four day interval using mercury column plethysmometer. [18]

Rheumatoid factor:

The latex turbidimetry method was used in the present study using RF turbilatex kit of SPINREACT Company. Calibration was carried out for linear range up to 100 IU/ml. The reading of RF factor of all the groups obtained was compared with the control animals and was expressed as IU/ml RF. [19]


Female wistar rats were sacrificed on 45th day of collagen administration and legs were removed and placed on formalin containing plastic bag. This plastic bag was kept at a distance of 90 cm from the X-ray source was and Radiographic analysis of arthritic and treated animal hind paw were performed by X-ray machine with a 300-mA exposition for 0.01 s. An investigator blinded for the treatment regimen performed radiograph score. The following radiograph criteria were considered: These scores (destroyed or intact joint) were used as a quantal test for bone necrosis. Radiographs were carefully examined using a stereo microscope and abnormalities were graded as follows:

(i) Periosteaic reaction, 0 - 3 (None, Slight, Moderate, Marked);

(ii) Erosions, 0 - 3 (None, Few, Many Small, Many Large);

(iii) Joint space narrowing, 0 - 3 (None, Minimal, Moderate, Marked);

(iv) Joint space destruction, 0 - 3 (None, Minimal, Extensive, Ankylosis).

Bone destruction was scored on the patella as described previously. [20]
Histological processing and assessment of arthritis damage:

Rats were killed by ether anesthesia. Knee joints were removed and fixed for 4 days in 4% formaldehyde. After decalcification in 5 % formic acid, the specimens were processed for paraffin embedding tissue sections (7 μm thick) and were stained with haematoxylin and eosin, or safranin. An experienced pathologist, unaware of the different drug treatments scored the condition of tibiotarsal joints. Histopathological changes were scored using the following parameters. Infiltration of cells was scored on a scale from 0 to 3, depending on the amount of inflammatory cells in the synovial tissues. Inflammatory cells in the joint cavity were graded on a scale from 0 to 3 and expressed as exudate. A characteristic parameter in arthritis is the progressive loss of articular cartilage. This destruction was separately graded on a scale from 0 to 3, ranging from the appearance of dead chondrocytes (empty lacunae) to complete loss of the articular cartilage. Bone erosion was scored on a scale ranging from 0 to 3, ranging from no abnormalities to complete loss of cortical and trabecular bone of the femoral head. Cartilage and bone destruction by pannus formation was scored ranging from 0, no change; 1, mild change (pannus invasion within cartilage); 2, moderate change (pannus invasion into cartilage/subchondral bone); 3, severe change (pannus invasion into the subchondral bone); and vascularity (0, almost no blood vessels; 1, a few blood vessels; 2, some blood vessels; 3, many blood vessels). Histopathological changes in the knee joints were scored in the femur region on 5 semiserial sections of the joint, spaced 70 μm apart. Scoring was performed on decoded slides by two observers, as described earlier. [20-23]


Following the primary and booster injections of CII/IFA, rats developed arthritis from day 21st onwards, and treatment with MPA, Dexamethazone(1 mg/kg), and vehicle were initiated on day 21st after the primary immunization and continued until day 45th of the experiment. As shown in Fig. 1, even after the onset of arthritis, MPA at high doses (200, 400 and 600 mg/kg) did markedly reduce hind paw volume of the arthritic rats in a dose-dependent manner as compared to the vehicle-treated arthritic rats.

Arthritic index and rheumatoid factor were significantly decreased in treatment with MPA (200 mg/kg, 400 mg/kg and 600mg/kg) and dexamethasone (1 mg/kg) treated animal as compare to disease control treatment.
Bone destruction, which is a common feature of arthritis, was examined by radiological analysis. Collagen administered rats had developed definite joint space narrowing of the intertarsal joints, diffuse soft tissue swelling that included the digits, diffuse demineralization of bone, marked periosteal thickening, and cystic enlargement of bone and extensive erosions produced narrowing or pseudowidening of all joint spaces. In contrast, in rats treated with MPA attenuate abnormalities consisted of asymmetric soft tissue swelling and small erosions, periosteal thickening, and minimal joint space narrowing, predominantly localized to the proximal areas of the paws.
As shown in Fig 5 a [NC]: Histology of synovial joint of normal control rat with intact morphology of synovium and synovial lining Fig 5 b [DC]: CA induced disease control rat showed plenty of lymphocytic infiltration [↓]in synovial lining with severe inflammation and marked angiogenesis [↕] studied with proliferation of synovial cells [↓↓]Fig 5 c [DEXA]: Dexamethasone [5 mg/kg] treated rats showed significant protection with mild lymphocytic infiltration [↓] with no evidence of thickening of synovial lining and angiogenesis Fig 5 d MPA200: MPA 200 treated rats showed milder angiogenesis [↕], lymphocytic infiltration [↓] and synovial lining thickening[↓↓]. Fig 5 e MPA400: MPA400 treated rats showed milder angiogenesis [↕], synovial lining thickening [↓↓] with no evidence of lymphocytic infiltration. Fig 5 f MPA600: MPA600 treated rats showed milder lymphocytic infiltration [↓] and synovial-lining thickening [↓↓] with no evidence of angiogenesis.

RA is a complicated refractory autoimmune disease characterized by a number of the inflammatory and destructive events such as joint pain and swelling, synovial hyperplasia,

pannus formation, cartilage and bone erosions, joint malformation.[24] Although various therapeutic approaches, such as cytokine-, receptor-, signal-transduction- or cell-type directed intervention, have been done, the inflammatory and destructive processes of RA are impeded partly, but rarely blocked fully, which reflects both the complexity and multiplicity

of the pathogenesis. These findings indicate that treatment targeting individual molecules might not suffice to stop the ongoing events, while combination therapy, which interferes with multiple targets, might be more effective for the treatment of RA.[25] In this regard, herbal medicines, comprising multiple active components with broad ranging pharmacological activities, represent an ancient model of combination therapy that could serve us well today.

Evaluation of the inflammatory stratus in RA is reflected by inflammation in the hind paw. Progression of disease in MPA treated group shows reduction in edema in dose dependent manner as compare to disease control animals. Symmetric involvement of small hand joints (especially proximal interphalangeal and metacarpophalangeal), foot joints (metatarsophalangeal), wrists, elbows, and ankles is typical, but initial manifestations may occur in any joint. Inflammation and / or nodules are observed on ears, nose, and tail, fore paws and hind paws. Arthritic index is the average of the score given to severity of the lesions in these places. This gives full picture of the disease. [26] MPA treated animal showed significant lesser arthritic index as compared with disease control animals.
Prominent immunological abnormalities that may be important in pathogenesis of RA include immune complexes are found in joint fluid cells and in vasculitis. Plasma cells produce antibodies e.g., rheumatoid factor (RF) that contribute to these complexes. Serum rheumatoid factor (RF) is the immunological expression of an individual's immune system reaction to the presence of an immunoglobulin molecule that is recognized as "non-self." This response to the "non-self" immunoglobulin results in the presence of immune complexes. These, in turn, bind complement and may eventually lead to synovium, cartilage, and bone destruction. Higher the levels of serum rheumatoid factor, higher are the development of inflammation.[27] Serum rheumatoid factor (RF) measures the amount of antibody IgM titer present in the serum.[28] MPA treated animal showed significantly lesser serum RF when compared to disease control animals.
Bone destruction, which is a common feature of arthritis, was examined by radiological analysis. Collagen treated rats had developed definite joint space narrowing of the intertarsal joints, diffuse soft tissue swelling that included the digits, diffuse demineralization of bone, marked periosteal thickening, and cystic enlargement of bone and extensive erosions produced narrowing or pseudowidening of all joint spaces. Despite a similar clinical course of arthritis, disease control rats suffered from more pronounced bone destruction than MPA treated group.
RA is characterized by synovial tissue leukocyte ingress and angiogenesis.[29] The disease is thought to occur as an immunological response to as yet unidentified antigen. Even in early RA, some of the earliest histological observations are blood vessels.[30] A mononuclear infiltrate characterizes the synovial tissue along with a luxuriant vasculature. Angiogenesis is integral to formation of the inflammatory pannus and without angiogenesis, leukocyte ingress could not occur. Changes in the density of blood vessels in the synovium and alterations in endothelial proliferative responses in RA have been shown in a range of studies. The number of synovial blood vessels has been found to correlate with hyperplasia of synovial cells, infiltration of mononuclear cells, and indices of joint tenderness.[31] Histopathology study of synovial joint showed that treatment with Parkinsonia aculeata Group decreased vascularity, lymphocytic infiltration with less rheumatoid inflammation and angiogenesis, with no thickening of synovial membrane and absence of lymphoid follicles. As compared to disease control and orally treated animals.

Our data suggested that Parkinsonia aculeata possesses significant antiarthritic activity. The possible mode of anti- arthritic activity of Methanolic extract of Parkinsonia aculeata appears to be, Possessing anti–inflammatory activity showed in arthritic parameters like Paw edema, Arthritic index, Rheumatoid factor, improving bone erosion.


The authors are thankful to the Director, Principal and management of C U Shah College of Pharmacy and Research, Wadhwan for providing necessary facilities to carry out this work. Our

acknowledgement to Dr. H A Solanki for their technical assistance in the plant collection and



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Fig. 1: Effect of Methanolic extract of Parkinsonia aculeata on paw edema in collagen induced arthritis in rat. Data are presented as Mean ± SEM (n=6), # P< 0.05, when compared with normal control; * P < 0.05, when compared with Disease control. (One Way ANOVA).

Fig. 2A

Fig. 2B

Fig. 2: A & B Effect of Methanolic extract of Parkinsonia aculeata on arthritic index and rheumatoid factor in collagen induced arthritis in rat. Data are presented as Mean ± SEM (n=6), @ P < 0.001, when compared with normal control, # P < 0.001, when compared with disease control, b P < 0.05, when compared with disease control (One Way ANOVA)

Fig. 3: Effect of Methanolic extract of Parkinsonia aculeata on radiographic score in collagen induced arthritis in rat. Data are presented as Mean ± SEM (n=6), @ P < 0.001, # P < 0.05 when compared with disease control, (One Way ANOVA)

Fig. 4: Effect of Methanolic extract of Parkinsonia aculeata on Radiographs of tibiotarsal joints

Fig. 5: Describe histopathology of joints indicate treatment with methanolic extract of Parkinsonia aculeata prevent bone erosion.

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